Hippocampal Microglia Activation Induced by Acute Pancreatic Injury in Rats.

BACKGROUND: Acute pancreatitis is an inflammation of the pancreatic glandular parenchyma that causes injury with or without the destruction of pancreatic acini. Clinical and experimental evidence suggest that certain systemic proinflammatory mediators may be responsible for initiating the fundamental mechanisms involved in microglial reactivity. Here, we investigated the possible repercussions of acute pancreatitis (AP) on the production of inflammatory mediators in the brain parenchyma focusing on microglial activation in the hippocampus. METHODS: The acute pancreatic injury in rats was induced by a pancreas ligation surgical procedure (PLSP) on the splenic lobe, which corresponds to approximately 10% of total mass of the pancreas. Blood samples were collected via intracardiac puncture for the measurement of serum amylase. After euthanasia, frozen or paraffin-embedded brains and pancreas were analyzed using qRT-PCR or immunohistochemistry, respectively. RESULTS: Immunohistochemistry assays showed a large number of Iba1 and PU.1-positive cells in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus of the PLSP group. TNF-α mRNA expression was significantly higher in the brain from PLSP group. NLRP3 inflammasome expression was found to be significantly increased in the pancreas and brain of rats of the PLSP group. High levels of BNDF mRNA were found in the rat brain of PLSP group. In contrast, NGF mRNA levels were significantly higher in the control group versus PLSP group. CONCLUSION: Our findings suggest that AP has the potential to induce morphological changes in microglia consistent with an activated phenotype.

Critical Frequency of Self-Heating in a Superelastic Ni-Ti Belleville Spring: Experimental Characterization and Numerical Simulation.

The mechanical loading frequency affects the functional properties of shape memory alloys (SMA). Thus, it is crucial to study its effect for the successful use of these materials in dynamic applications. Based on the superelastic cyclic behavior, this work presents an experimental methodology for the determination of the critical frequency of the self-heating of a NiTi Belleville conical spring. For this, cyclic compressive tests were carried out using a universal testing machine with loading frequencies ranging from 0.5 Hz to 10 Hz. The temperature variation during the cyclic tests was monitored using a micro thermocouple glued to the NiTi Belleville spring. Numerical simulations of the spring under quasi-static loadings were performed to assist the analysis. From the experimental methodology applied to the Belleville spring, a self-heating frequency of 1.7 Hz was identified. The self-heating is caused by the latent heat accumulation generated by successive cycles of stress-induced phase transformation in the material. At 2.0 Hz, an increase of 1.2 °C in the average temperature of the SMA device was verified between 1st and 128th superelastic cycles. At 10 Hz, the average temperature increase reached 7.9 °C and caused a 10% increase in the stiffness and 25% decrease in the viscous damping factor. Finally, predicted results of the force as a function of the loading frequency were obtained.

Continuous Loading of Ultracold Ground-State

We have developed an approach to continuously load ultracold ^{85}Rb_{2} vibrational ground-state molecules into a crossed optical dipole trap from a magneto-optical trap. The technique relies on a single high-power light beam with a broad spectrum superimposed onto a narrow peak at an energy of about 9400 cm^{-1}. This single laser source performs all the required steps: the short-range photoassociation creating ground-state molecules after radiative emission, the cooling of the molecular vibrational population down to the lowest vibrational level v_{X}=0, and the optical trapping of these molecules. Furthermore, we probe by depletion spectroscopy and determine that 75% of the v_{X}=0 ground-state molecules are in the three lowest rotational levels J_{X}=0, 1, 2. The lifetime of the ultracold molecules in the optical dipole trap is limited to about 70 ms by off-resonant light scattering. The proposed technique opens perspectives for the formation of new molecular species in the ultracold domain, which are not yet accessible by well-established approaches.

Quantitative assessment of DNA damage in the industrial ethanol production strain Saccharomyces cerevisiae PE-2.

Lignocellulosic hydrolysates remain one of the most abundantly used substrates for the sustainable production of second generation fuels and chemicals with Saccharomyces cerevisiae. Nevertheless, fermentation inhibitors such as acetic acid, furfural and hydroxymethylfurfural are formed during the process and can lead to slow or stuck fermentations and/or act as genotoxic agents leading to production strain genetic instability. We have developed a novel dominant deletion (DEL) cassette assay for quantification of DNA damage in both wild-type and industrial yeast strains. Using this assay, the ethanol production strain S. cerevisiae PE-2 was shown to be more resistant to hydrogen peroxide and furfural than the laboratory DEL strain RS112. Indeed, the PE-2 strain also showed a lower tendency for recombination, consistent with a more efficient DNA protection. The dominant DEL assay presented herein should prove to be a useful tool in the selection of robust yeast strains and process conditions for second generation feedstock fermentations.

Beckwith-Wiedemann syndrome mimicking the classical form of congenital adrenal hyperplasia in newborn screening.

Beckwith-Wiedemann syndrome (BWS) is a common genetic congenital disease characterized by somatic overgrowth and its broad clinical spectrum includes pre- and post-natal macrosomia, macroglossia, visceromegaly, increased risk of neonatal hypoglycemia, and development of embryonic tumors. BWS occurs due to genetic/epigenetic changes involving growth-regulating genes, located on region 11p15, with an important genotype-phenotype correlation. Congenital adrenal hyperplasia (CAH) comprises a spectrum of autosomal recessive diseases presenting a variety of clinical manifestations due to a deficiency in one of the enzymes involved in cortisol secretion. Early diagnosis based on newborn screening prevents the adrenal crisis and early infant death. However, high 17-hydroxyprogesterone (17-OHP) levels can occur in newborns or premature infants without CAH, in situations of stress due to maternal or neonatal factors. Here, we report new cases of false-positive diagnosis of 21-hydroxylase deficiency during newborn screening - two girls and one boy with BWS. Methylation-specific multiplex ligation-dependent probe amplification revealed a gain of methylation in the H19 differentially methylated region. Notably, all three cases showed a complete normalization of biochemical changes, highlighting the transient nature of these hormonal findings that imitate the classical form of CAH. This report sheds light on a new cause of false-positive 21-hydroxylase deficiency diagnosis during newborn screening: Beckwith-Wiedemann syndrome.

Bacteria and Yeast Colony PCR.

The bacteria Escherichia coli and the yeast Saccharomyces cerevisiae are currently the two most important organisms in synthetic biology. E. coli is almost always used for fundamental DNA manipulation, while yeast is the simplest host system for studying eukaryotic gene expression and performing large-scale DNA assembly. Yeast expression studies may also require altering the chromosomal DNA by homologous recombination. All these studies require the verification of the expected DNA sequence, and the fastest method of screening is colony PCR, which is direct PCR of DNA in cells without prior DNA purification. Colony PCR is hampered by the difficulty of releasing DNA into the PCR mix and by the presence of PCR inhibitors. We hereby present one protocol for E. coli and two protocols for S. cerevisiae differing in efficiency and complexity as well as an overview of past and possible future developments of efficient S. cerevisiae colony PCR protocols.

Gastrotomy followed by gastrorrhaphy as a reliable and more physiologic technique for inducing peritoneal adhesion in rats.

OBJECTIVE: this research objective was to develop a new peritoneal adhesion animal model that would lead to adhesions formation in all operated animals, simple and reproducible, associated with maintenance the animal's health. METHODS: eighteen adult male Wistar rats (Rattus norvegicus) were randomly distributed into three groups: Control Group (anatomical and clinical parameters), Sham Group (delicate manipulation of the stomach and exposure of the peritoneal cavity to ambient air) and Surgery Group (gastrotomy followed by gastrorrhaphy). The animals were analyzed and classificated macroscopically according to two adhesion classification models and differences between groups were considered significant when p<0.05. RESULTS: the six animals in the control group had no peritoneal adhesions, three of the six animals in the sham group had focal peritoneal adhesions, and all animals in the surgery group (gastrotomy followed by gastrorraphy) had firm peritoneal adhesions. All adhesions found were macroscopically quantified and microscopically confirmed, without carrying out a microscopic classification of the adhesions. CONCLUSION: the new model developed of gastrotomy followed by gastrorrhaphy, proved to be safe and efficient to induce and study peritoneal adhesions.

Optimization of a hybrid bacterial/Arabidopsis thaliana fatty acid synthase system II in Saccharomyces cerevisiae.

Fatty acids are produced by eukaryotes like baker's yeast Saccharomyces cerevisiae mainly using a large multifunctional type I fatty acid synthase (FASI) where seven catalytic steps and a carrier domain are shared between one or two protein subunits. While this system may offer efficiency in catalysis, only a narrow range of fatty acids are produced. Prokaryotes, chloroplasts and mitochondria rely instead on a FAS type II (FASII) where each catalytic step is carried out by a monofunctional enzyme encoded by a separate gene. FASII is more flexible and capable of producing a wider range of fatty acid structures, such as the direct production of unsaturated fatty acids. An efficient FASII in the preferred industrial organism S. cerevisiae could provide a platform for developing sustainable production of specialized fatty acids. We functionally replaced either yeast FASI genes (FAS1 or FAS2) with a FASII consisting of nine genes from Escherichia coli (acpP, acpS and fab -A, -B, -D, -F, -G, -H, -Z) as well as three from Arabidopsis thaliana (MOD1, FATA1 and FATB). The genes were expressed from an autonomously replicating multicopy vector assembled using the Yeast Pathway Kit for in-vivo assembly in yeast. Two rounds of adaptation led to a strain with a maximum growth rate (µmax) of 0.19 h-1 without exogenous fatty acids, twice the growth rate previously reported for a comparable strain. Additional copies of the MOD1 or fabH genes resulted in cultures with higher final cell densities and three times higher lipid content compared to the control.

Heterologous mesenchymal stem cells successfully treat femoral pseudarthrosis in rats.

BACKGROUND: This study evaluated the effectiveness of treating pseudarthrosis in rats by using bone marrow cell suspensions or cultures of bone marrow mesenchymal stromal cells METHODS: Thirty-eight specific pathogen-free (SPF) animals were randomly assigned to four groups: Group 1, Control, without surgical intervention; Group 2 (Placebo), experimental model of femoral pseudarthrosis treated only with saline solution; Group 3, experimental model of femoral pseudarthrosis treated with heterologous bone marrow cells suspension; Group 4, experimental model of femoral pseudarthrosis treated with cultures of heterologous mesenchymal stromal cells from bone marrow. When pseudarthrosis was confirmed by simple radiological studies, digital radiography and histopathology after a 120-day postoperative period, Groups 2, 3 and 4 were treated as above. At 30, 60 and 90 days after the treatment, all animals were evaluated by simple radiological studies, and at the end of the experiment, the animals were assessed by computed axial tomography and anatomopathological and histomorphometric examinations. RESULTS: Injected cells were detected in the areas affected by pseudarthrosis using scintigraphy within the first 24 hours after their administration. After 60 days, the animals of Group 3 showed callus formation while the animals of Group 4 presented periosteal reaction and had some consolidated areas. In contrast, Group 2 showed a predominance of fibro-osteoid tissue. After 90 days, bone consolidation and remodeling was observed in all animals from Group 3 whereas animals from Group 4 exhibited partial consolidation and those ones from Group 2 persisted with pseudarthrosis. CONCLUSION: The treatment with heterologous bone marrow cells suspension proved to be effective in the treatment of pseudarthrosis whereas cultures of heterologous bone marrow mesenchymal stromal cells did not show the same potential to aid bone healing.

Downregulation of C3 and C4A/B complement factor fragments in plasma from patients with squamous cell carcinoma of the penis.

PURPOSE: To investigate the use of ClinProt technique to identify cancer markers in plasma of patients suffering from squamous cell carcinoma of the penis (SCCP). MATERIALS AND METHODS: Plasma of 36 healthy subjects and 25 patients with penile carcinoma who underwent surgical treatment between June 2010 and June 2011 was collected and analyzed by the ClinProt/MALDI/ToF technique. Then the peptides were identified from the C8 MB eluted fraction of patients' and control subjects' plasma by LIFT MS/MS. RESULTS: A cluster of 2 peptides (A=m/z 1897.22 ± 9 Da and B=m/z 2021.99 ± 9 Da) was able to discriminate patients from control subjects. Cross validation analysis using the whole casuistic showed 62.5 % and 86.76 % sensitivity and specificity, respectively. The cluster also showed very high sensitivity (100 %) and specificity (97%) for SCCP patients that died due to the disease. Furthermore, patients with lymph node involvement presented sensitivity and specificity of 80 % and 97 %, respectively. These two peptides were identified by the proteomic approach based on a MALDI-TOF/TOF as fragments of C3 (m/z 1896.17) and C4a/b (m/z 2021.26) complement proteins. CONCLUSIONS: The results showed that as the disease progresses, the fragments C3 and C4 A/B are less expressed in comparison with healthy subjects. These results may be useful as prognostic tools.

Three new species of the Neurigona orbicularis species group from Brazil (Diptera: Dolichopodidae).

The Neurigona orbicularis species group (Diptera: Dolichopodidae) is recorded from Brazil for the first time, and three new species are described and illustrated, including figures of the male genitalia: N. lopesi sp. nov. (Tocantins and Gois, Cerrado biome), N. papaveroi sp. nov. (Mato Grosso, Cerrado biome) and N. tingua sp. nov. (Rio de Janeiro, Atlantic Forest biome). Additionally, N. orbicularis Becker, previously known only from the type-locality in Paraguay, is newly recorded from the Brazilian States of Mato Grosso do Sul (Pantanal biome) and So Paulo (Atlantic Forest biome) and its hypopygium is also illustrated. An updated identification key to males of the orbicularis-group is provided.

Glyphosate-Based Herbicide Causes Cellular Alterations to Gut Epithelium of the Neotropical Stingless Bee Melipona quadrifasciata quadrifasciata (Hymenoptera: Meliponini).

Glyphosate-based herbicides (GBH) are the best-selling pesticides in Brazil, with hundreds of thousands of tons sold per year. There is no study investigating morphological alterations caused by GBH on the epithelium of the gut in bees. Here, we aimed to demonstrate effects of chronic ingestion of GBH in the midgut digestive cells of the Brazilian stingless bee Melipona quadrifasciata quadrifasciata Lepeletier 1836. We kept forager workers of M. quadrifasciata in laboratory conditions and fed on food contaminated with three different concentrations of GBH for 10 days, after which the midgut digestive cell structure and ultrastructure were analyzed. The presence of GBH in food did not affect food consumption, indicating that M. quadrifasciata bees do not reject food contaminated with GBH. As digestive cells of the midgut release apocrine secretion as a detoxication mechanism, we expected that the ingestion of food contaminated with GBH in the present study affect the height of midgut digestive cells. However, such reduction did not occur, probably because of the low-test concentrations. Although there were differences in digestive cell ultrastructure, ingestion of GBH impaired midgut digestive cell cohesion by disorganizing the smooth septate junctions between cells, which may probably be caused by the adjuvant "polyethoxylated tallow amine" present in the GBH. Previous studies demonstrated that GBH increase bees' sensibility to intestine infections, based on the present results we hypothesized that the loss of cell cohesion in the midgut epithelium favors pathogenic microbial infections and harms food absorption, increasing bees' mortality.

Laparoscopic Pancreatectomy in Rats: The Development of an Experimental Model.

INTRODUCTION: several pancreatectomy techniques in rats have been described and utilized for research concerning the pancreas endocrine and exocrine functions. However, we did not find a description of any kind of laparoscopic pancreatectomy in rats in the consulted databases. The objective of this study is to describe a laparoscopic splenic lobe pancreatectomy in rats. METHODS: ten Wistars rats weighting more then 300 g were operated for standardizing the laparoscopic pancreatosplenectomy technique, aided by previous descriptions of laparoscopic splenectomy and open pancreatectomy in rats. Adjustments have been progressively adopted for technical refinement. RESULTS: In five animals a low-cost rat laparoscopic set was used. In other five animals we used a standard laparoscopic set. Three rats died early due to different causes: transection of the gastroesophageal junction, hemorrhagic shock and inadvertent colonic injury. The postoperative period of the other seven rats was uneventful until the seventh postoperative day. DISCUSSION: laparoscopic distal pancreatectomy and splenectomy in rats is feasible and safe, even with a low-cost set, in which the results were alike the standard laparoscopic set.

Enhanced Solubility of Albendazole in Cyclodextrin Inclusion Complex: A Molecular Modeling Approach and Physicochemical Evaluation.

BACKGROUND: Albendazole (ABZ) is the drug of choice for the treatment of a variety of human and veterinary parasites. However, it has low aqueous solubility and low bioavailability. Cyclodextrins (CD) are pharmaceutical excipients with the ability to modulate the solubilization property of hydrophobic molecules. OBJECTIVE: The aim of the study was to analyze through in vitro and in silico studies (Autodock Vina software and CycloMolder platform) the formation of inclusion complexes between ABZ, ß-cyclodextrin (ß-CD) and its derivatives Methyl-ß-cyclodextrin (M-ß-CD) and Hydroxypropyl-ß-cyclodextrin (HP-ß-CD). METHODS: The most stable inclusion complexes were produced by the kneading method and characterized by Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), X-Ray Diffraction (XRD), determination of the ABZ content and in vitro dissolution profile. RESULTS: Molecular modeling revealed that inclusion complexes between HP-ß-CD:ABZ (in the proportion 1:1 and 2:1) presented the lowest formation energy and the highest number of intermolecular interactions, showing that the use of more cyclodextrins does not generate gains in the stability of the complex. On the characterization tests, the complexes experimentally obtained by the kneading method demonstrated highly suggestive parameters, including ABZ in HP-ß-CD in both molar proportions, suppression of bands in the infrared spectrum, displacement of the drug's melting temperature in DSC, crystallinity halos instead of the characteristic peaks of ABZ crystals in the XRD and a release of more than 80% of ABZ in less than 5 minutes, dissolution efficiency of up to 92%. CONCLUSION: In silico studies provided a rational selection of the appropriate complexes of cyclodextrin, enabling the elaboration of more targeted complexes, decreasing time and costs for elaboration of new formulations, thereby increasing the oral biodisponibility of ABZ.

Reduction of synchronous reluctance motor currents with minimization of direct and cross saturation magnetic model.

In this paper, two cost functions are presented which are minimized to provide the optimized reference currents for the controlled drive of the synchronous reluctance motor. Since the synchronous reluctance motor operation is divided into two operating regions: one up to rated speed (0-1740 rpm), known as constant torque region and variable power, and other above this speed, called field weakening region (1740-2000 rpm), each of the cost functions proposals is appropriate for a specific region. The optimization problem is solved by the method of Lagrange Multiplier which allows finding the minimum value of a function with one or more constraints. The tests show a reduction in the motor current value of around 2%.

A novel D-xylose isomerase from the gut of the wood feeding beetle Odontotaenius disjunctus efficiently expressed in Saccharomyces cerevisiae.

Carbohydrate rich substrates such as lignocellulosic hydrolysates remain one of the primary sources of potentially renewable fuel and bulk chemicals. The pentose sugar D-xylose is often present in significant amounts along with hexoses. Saccharomyces cerevisiae can acquire the ability to metabolize D-xylose through expression of heterologous D-xylose isomerase (XI). This enzyme is notoriously difficult to express in S. cerevisiae and only fourteen XIs have been reported to be active so far. We cloned a new D-xylose isomerase derived from microorganisms in the gut of the wood-feeding beetle Odontotaenius disjunctus. Although somewhat homologous to the XI from Piromyces sp. E2, the new gene was identified as bacterial in origin and the host as a Parabacteroides sp. Expression of the new XI in S. cerevisiae resulted in faster aerobic growth than the XI from Piromyces on D-xylose media. The D-xylose isomerization rate conferred by the new XI was also 72% higher, while absolute xylitol production was identical in both strains. Interestingly, increasing concentrations of xylitol (up to 8 g L-1) appeared not to inhibit D-xylose consumption. The newly described XI displayed 2.6 times higher specific activity, 37% lower KM for D-xylose, and exhibited higher activity over a broader temperature range, retaining 51% of maximal activity at 30 °C compared with only 29% activity for the Piromyces XI.

Impact of the reduction in TSH cutoff level to 6 mIU/L in neonatal screening for congenital hypothyroidism in Santa Catarina: final results.

OBJECTIVE: To assess the implications of changing the cutoff level of TSH from 10 to 6 mIU/L. METHODS: The study population was constituted by 74.123 children screened for congenital hypothyroidism by the National Screening Program in Santa Catarina, from March 2011 to February 2012. The cutoff of TSH was 6 mIU/L. If TSH between 6-10 mIU/L, the newborn was recalled for a second TSH measurement on filter paper. If TSH > 6 mIU/L in the second sample, the child was sent for medical evaluation. In children with normal topic thyroid, levothyroxine was suspended for 1 month at the age of 3 years for identification of the etiology and evaluation of the need to continue treatment. RESULTS: Among the children screened, 435 were recalled for presenting TSH between 6 and 10 mIU/L in the first sample, 28 remained TSH > 6 mIU/L in the second sample. Among these, 11 had a final diagnosis of dyshormonogenesis, two of ectopic thyroid, two of thyroid hypoplasia and one of transient hypothyroidism. Ten children presented normal TSH levels on the first medical evaluation and two lost follow-up. CONCLUSION: A decrease in the TSH cutoff level from 10 to 6 mIU/L in a neonatal screening program for congenital hypothyroidism reduced the number of false-negative results, increasing the sensitivity of the test, but increased the number of false-positive results and recalls. Since a TSH cutoff level of 6 mIU/L detects thyroid function abnormalities requiring treatment, the adoption of this cutoff level is justified.

Cryopreserved Rat Thyroid Autotransplantation in the Treatment of Postoperative Hypothyroidism.

To verify the viability and functionality of cryopreserved thyroid autotransplantation in rats who underwent total thyroidectomy in the treatment of postoperative hypothyroidism. Thirty-two Wistar rats were randomly assigned into groups (G) with eight animals each: control (CG); simulation (SG); hypothyroidism (HTG) and transplanted (TG). At the beginning and in the 13th week of the experiment, serum levels of total T3, free T4, TSH and calcium were determined. In both the first and 14th weeks, scintigraphic examinations, 99m-Tc pertechnetate radioisotope biodistribution and histopathology were performed. In the 14th week, the expression of proliferating cell nuclear antigen (PCNA) and cellular apoptosis (caspase-3) were also evaluated. In the 13th week, the transplanted animals had normal serum levels of total T3 and free T4. TSH levels showed a tendency towards normality. In the 14th week, scintigraphic exams displayed graft isotopic uptake in all animals in the TG group. Histological examinations 13 weeks after transplantation showed the viability and functionality of thyroid follicles. PCNA revealed significant immunoreactivity of the graft (p < 0.001) when the TG was compared to the CG. There was no difference between CG and TG considering the expression of activated caspase-3. The experimental study confirmed the viability and functionality of thyroid autotransplantation implanted in skeletal muscle with evidence of cell proliferation without cellular apoptosis. This surgical strategy was effective in the treatment of postoperative hypothyroidism.

Epidemic spreading with awareness and different timescales in multiplex networks.

One of the major issues in theoretical modeling of epidemic spreading is the development of methods to control the transmission of an infectious agent. Human behavior plays a fundamental role in the spreading dynamics and can be used to stop a disease from spreading or to reduce its burden, as individuals aware of the presence of a disease can take measures to reduce their exposure to contagion. In this paper, we propose a mathematical model for the spread of diseases with awareness in complex networks. Unlike previous models, the information is propagated following a generalized Maki-Thompson rumor model. Flexibility on the timescale between information and disease spreading is also included. We verify that the velocity characterizing the diffusion of information awareness greatly influences the disease prevalence. We also show that a reduction in the fraction of unaware individuals does not always imply a decrease of the prevalence, as the relative timescale between disease and awareness spreading plays a crucial role in the systems' dynamics. This result is shown to be independent of the network topology. We finally calculate the epidemic threshold of our model, and show that it does not depend on the relative timescale. Our results provide a new view on how information influence disease spreading and can be used for the development of more efficient methods for disease control.

Beckwith-Wiedemann syndrome mimicking the classical form of congenital adrenal hyperplasia in newborn screening

SUMMARY Beckwith-Wiedemann syndrome (BWS) is a common genetic congenital disease characterized by somatic overgrowth and its broad clinical spectrum includes pre- and post-natal macrosomia, macroglossia, visceromegaly, increased risk of neonatal hypoglycemia, and development of embryonic tumors. BWS occurs due to genetic/epigenetic changes involving growth-regulating genes, located on region 11p15, with an important genotype-phenotype correlation. Congenital adrenal hyperplasia (CAH) comprises a spectrum of autosomal recessive diseases presenting a variety of clinical manifestations due to a deficiency in one of the enzymes involved in cortisol secretion. Early diagnosis based on newborn screening prevents the adrenal crisis and early infant death. However, high 17-hydroxyprogesterone (17-OHP) levels can occur in newborns or premature infants without CAH, in situations of stress due to maternal or neonatal factors. Here, we report new cases of false-positive diagnosis of 21-hydroxylase deficiency during newborn screening - two girls and one boy with BWS. Methylation-specific multiplex ligation-dependent probe amplification revealed a gain of methylation in the H19 differentially methylated region. Notably, all three cases showed a complete normalization of biochemical changes, highlighting the transient nature of these hormonal findings that imitate the classical form of CAH. This report sheds light on a new cause of false-positive 21-hydroxylase deficiency diagnosis during newborn screening: Beckwith-Wiedemann syndrome.